Plants carrying one or more expressible heterologous genes have a variety of potential advantages. The plants carrying a gene expression cassette may carry one or more genes which confer desired traits, including for example, herbicide, pesticide or insect tolerance; tolerance to stress; enhanced flavor and/or shelf life of the fresh produce (fruit, vegetable, seeds etc.), as well as the ability to amplify the synthesis of useful plant endogenous and/or foreign proteins, sugars, fatty-acids or secondary metabolites for consumption by man and/or animals or for use as raw materials in a variety of industries (cosmetics, pharmaceuticals, nutraceutics, foods, paper, fibers, etc.).
Current transformation technologies provide an opportunity to engineer plants with desired traits, and major advances in plant transformation have been occurred in recent years. The most common technology is mediated by Agrobacterium tumefaciens. Other methods of transformation are direct DNA transfer including microinjection, electroporation, particles bombardment and viral vectors (Birch R G. 1997. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:297-326). With the exception of viral vectors, application of the above techniques results in the penetration of foreign DNA into the treated cells/tissue, its integration into the treated plant's genome and the regeneration of transgenic plants. However, in many major crop plants, serious genotype limitations still exist. Transformation of some agriculturally important crop plants continues to be both difficult and time consuming. In addition, in all the aforementioned technologies, transformation is conducted with vegetative tissues (including embryos) the efficiency of transformation is poor, markers for selection are required, and the percentage of successful regeneration of a plant from the transformed cell or tissue is rather low.
Integration of the foreign DNA into the plant genome is not always desirable, particularly when genetically modified (GMO) plants arouse environmental and political issues. In these cases, methods enabling the expression of the heterologous gene(s) that are not incorporated into the host genome are required.
International (PCT) Application Publication No. WO 2007/141790 to some of the inventors of the present invention discloses modified Geminivirus-based constructs capable of spreading from one plant cell to the other within the treated plant and concomitantly introducing foreign DNA into the plant cells, without exerting the pathological virus symptoms. This foreign DNA is expressed in the plant tissues but is not integrated into its genome. The constructs (also referred to as expression vectors, IL-60 being one example) comprise the heterologous polynucleotide sequence that interrupts the Geminivirus replicase genes such that it is flanked by a non-contiguous nucleic acid sequence encoding a Geminivirus replicase or replicase-associated protein.
Seed priming is a process for treating plant seeds that enables them to undergo faster and more uniform germination on sowing or planting compared to non-treated seeds. Additionally, priming offers an optional simultaneous treatment with fungicide/pesticides/fertilizers or other chemicals (e.g. coating, pelleting, coloring as a trade mark, and more) providing protection and/or facilitating germination, emergence and seedlings establishment.
Priming allows the seeds to absorb enough water to enable their pre-germinative metabolic processes to begin and then arrests them at that stage. The amount of water absorbed (with or without beneficial chemicals) must be carefully controlled, as too much would simply allow the seed to germinate and too little would result in the seed ageing. Once the correct amount of water has been absorbed, the seeds may be sown or dried back to the original water content for storage. The primed seeds usually germinate and emerge more quickly and uniformly and the seedling vigor is typically higher compared to unprimed seeds, particularly under suboptimal conditions. The benefits gained by priming, such as rate and uniformity of germination are usually attributed to the initiation of DNA-repair processes, protein hydration enzyme activation and additional processes occurring in the early stages of germination.
The three major techniques used for controlled water uptake include priming with aqueous solutions, with hydrated solid particulate systems or by controlled hydration with water. Priming with solution is based on immersing the seeds in osmotic solution, typically PEG solutions characterized by osmotic potential that enables limited imbibition that is insufficient for full hydration and seed germination. Alternatively, the same effect is achieved by mixing the seeds with hydrated absorbent medium such as clay or peat (e.g., U.S. Pat. No. 4,912,874). Controlled hydration with aqueous solutions may be achieved, for example, by utilizing semi-permeable membrane to mediate the transfer of water from a solution of a given osmotic pressure to the seed (U.S. Pat. No. 5,992,091). Priming is performed under a variety of temperatures and aeration methods (e.g., stirring, agitation, bubbling, etc.) using any of the techniques for controlled water uptake (Taylor A G. et al. 1998. Seed Science Technology 8:245-256).
Combination between the common transformation techniques described hereinabove and seed priming has been disclosed. For example, U.S. Pat. No. 6,646,181 discloses a method of introducing genes into plants comprising synchronizing the stage of development of the plant at a stage that includes large amounts of 4C DNA in seeds of the plants and transfecting the cells of the seeds, in which synchronizing the stage of development comprises admixing a particulate solid matrix material and a seed priming amount of water, with aeration of seeds, for a time and at a temperature sufficient to cause a substantial number of the cells of the seeds to reach a desired stage of a cell cycle.
U.S. Patent Application Publication No. 2006/0005273 discloses maize explants suitable for transformation. The explants comprise a maize seed split in half longitudinally, wherein the splitting exposes the scutellum, the coleoptilar ring and shoot apical meristem, each of which are independently suitable for transformation. Priming the seed prior to splitting with either callus or shoot priming medium increases the callus and shoot induction frequency after transformation.
U.S. patent application Ser. No. 20100154083 discloses compositions and methods to screen, identify, select, isolate, and/or regenerate targeted integration events using seed priming. Seed priming provides the identification of a seed having stably incorporated into its genome a site-specific recombinase mediated integration of a selectable marker at a target locus operably linked to a promoter active in the seed.
These combination methods employ the known methods of transformation and are aimed at increasing the efficacy of foreign gene integration into the plant genome. As described hereinabove, the available methods have limitations with regard to applicability and efficacy. In addition integration of the foreign DNA to the plant genome is not always desirable.
There is a recognized need for, and it would be highly advantageous to have means and methods for simple and efficient introduction of DNA into plant cells that further enable efficient plant regeneration.